Project Summary This proposal focuses on tissue resident memory T cells (TRM) that are essential for protection of the mucosa against reinfection. Infectious and parasitic diseases cause more than 130,000 deaths a year in the United States and preventive vaccines play a major role in reducing the burden of infectious diseases on human health. However, the different routes of infection and genetic variability of microbes pose great challenges to developing protective vaccines for several important pathogens such as influenza, HIV, and Mycobacterium tuberculosis. It is likely that in addition to generating neutralizing antibodies, the most efficacious vaccines will generate T cells that localize to the portals of pathogen entry and recognize conserved epitopes across viral strains. Thus, for a large number of mucosal infections, next generation of vaccines will require a better understanding of how T cell memory is generated in mucosal tissues, maintained and reactivated upon reinfection. TRM cells are a key component in the protection against viral and bacterial infections. Nearly every organ has specialized TRM cells that can provide local protection and in mucosal tissues, like lung and gut, the intraepithelial TRM cells lie directly at the frontline of pathogen entry. It is not clear how TRM cells differentiate and persist long-term in various tissues, but likely the genetic programs for each TRM cell population is built from a combination of common and tissue-specific signals. Indeed, mouse TRM cells have a common gene expression signature that contains NR4A1 and NR4A2. NR4A1 is a gene that we have found to be essential for the development and maintenance of CD8+ TRM in many tissues. NR4A1and NR4A2 are nuclear hormone receptors. These are transcription factors modulated by small molecules derived from metabolism, hormones or diet. NR4A1 and NR4A2 are orphan receptors, meaning that their ligands are unknown. The first aim of this application is to identify the metabolites that bind to NR4A1 and NR4A2. For this we will perform large scale immunoprecipitations of NR4A1 and NR4A2 from cell lines that have strong NR4A1 and NR4A2 specific activity and identify its ligands by high accuracy mass spectrometry. The second aim of this application is to investigate NR4A1 and NR4A2 activity in CD8+ TRM cells in vivo. We will develop a reporter system to track the activity of NR4A1 and NR4A2 in wild type cells as well as ?ligand deficient cells induced by a CRISPR based transcriptional repressor that inhibits the expression of the metabolic enzyme required for the synthesis of the NR4A1 or NR4A2 ligand in CD8+ T cells. We will transfer these cells to C57BL/6 mice and measure the effect of knockdown of NR4A1 or NR4A2 ligand biosynthetic enzyme on the differentiation of TEM, TCMs and TRMs after infection with LCMV. !